Activation of mature T lymphocytes is a multi-step process requiring both Ag-specific triggering of the TCR complex and co-stimulation mediated through the CD28-CD80/CD86 pathway. CTLA-4 is a critical inhibitor of T cell activation as evidenced by the lethal lympho-proliferation seen in CTLA-4 knockout mice. These signaling pathways play a primary role in T cell homeostasis and manipulating these pathways has emerged as a powerful strategy to suppress autoimmunity with clinical applications. In the past, Tolerogenics generated bispecific antibodies (BsAb) with specificities for TSHR and CTLA-4. The anti-TSHR portion of the BsAb could bind to the TSHR-expressing thyroid tissue leaving the anti-CTLA-4 portion to engage CTLA-4 expressed on the attacking T cells. Our results showed that using this BsAb tolerance could be induced and autoimmune thyroiditis suppressed. The disease suppression was associated with selective expansion of a subpopulation of CD4+CD25+ Treg cells. These results supported the notion that targeted CTLA-4 engagement could result in a selective expansion of Treg cells that can mediate persistent hyporesponsiveness to specific self antigens. Recently, Similar protective effects against Hashimoto's thyroiditis and type-1 diabetes have been noted using antigen-pulsed DCs coated with a CD11c (a dendritic cell surface marker) and CTLA-4 specific BsAb. Therefore, targeted engagement of CTLA-4 on activated T cells could provide an effective means of suppressing autoimmunity in NOD mouse model of type-1 diabetes. Based on these observations, the Company hypothesizes that "Engagement of CTLA-4 concomitant with Ag-specific T-Cell Receptor (TCR) ligation can be used to down modulate pathogenic self reactive T effector cell ("Teff") responses, while inducing Tregs that can suppress Teff function as a means of targeted therapy to: i) prevent the development of T1D;and, ii) stabilize or ameliorate ongoing T1D." To test this hypothesis, Tolerogenics will develop a BsAb that can selectively target mouse beta cells through GLUT2 and down modulate beta cell infiltrating T cells through targeted CTLA-4 engagement in non-obese diabetic (NOD) mice that spontaneously develop type-1 diabetes. Aim-1. Construction and characterization of BsAb to mouse GLUT2 and CTLA-4. Aim-2. To test the ability of anti-mouse GLUT2-anti-CTLA-4 BsAb to suppress T1D. Generation of BsAb that can be used to specifically suppress, stabilize or reverse type-1 diabetes will have significant clinical implications for developing novel therapies for type-1 diabetes in humans. PUBLIC HEALTH RELEVANCE: Autoimmune disease is the third major category of illnesses plaguing the United States and the most common of these diseases affect more than 8.5 million Americans. The burden of immune-mediated diseases is staggering. In the US alone, these conditions result in direct and indirect costs that exceed $100 billion. Existing clinical approaches to autoimmune disorders have relied on the administration of immunosuppressive drugs that result in suppressing the entire immune system. Such a response makes a patient susceptible to a wide range of infectious agents. In Tolerogenics'application, the company proposes to test specific therapeutic approaches aimed at restoring immune regulation and down regulating only the aberrant immune responses using a proprietary bispecific antibody for the treatment of Type I Diabetes, initially. This technology could have major importance not only Type I Diabetes treatment efforts, but for a variety of other autoimmune diseases in the United States including systemic lupus erythematosus (SLE), rheumatoid arthritis, multiple sclerosis, Grave's thyroid disease, Hashimoto's thyroiditis, and others.